Laboratory evaluation of rapid test kits to detect hepatitis C antibody for use in predonation screening in emergency settings.

BACKGROUND: Emergency whole blood transfusion is a lifesaving procedure employed on modern battlefields. Rapid device tests (RDTs) are frequently used to mitigate transfusion-transmitted infection risks. STUDY DESIGN AND METHODS: A limited evaluation of the RDT formerly used on battlefields was performed using 50 donor plasma samples and commercially available panels. Five hepatitis C virus (HCV) RDTs with sufficient stated sensitivity and thermostability were assessed using 335 HCV-positive and 339 HCV-negative donor plasma samples, 54 seroconversion panel plasma samples, and 84 HCV-positive and 84 HCV-negative spiked whole blood under normal, hot, and cold storage conditions and normal and hot test conditions, plus an ease-of-use survey. RESULTS: BioRapid HCV test sensitivity on donor plasma was 84% (95% confidence interval [CI], 70.9%-92.8%). Using all positive plasma samples, OraQuick HCV sensitivity exceeded all comparators (99.4%, 95% CI, 98.0%-99.9%, p<0.05). Specificity was consistently high, led by OraQuick HCV at 99.7% (95% CI, 98.6%-100%), statistically superior only to Axiom HCV (p<0.05). Using seroconversion panels, only OraQuick HCV showed equivalent or earlier HCV detection compared to the gold standard. Using spiked whole blood, specificity was consistently high, and sensitivity ranged significantly from 34.5% (95% CI, 25.0%-45.1%) for CORE HCV to 98.8% (95% CI, 94.3%-99.9%) for OraQuick HCV. All comparator RDTs were significantly less sensitive than OraQuick HCV at one or more stress condition. CONCLUSION: This HCV RDT comparison identified significant sensitivity differences, particularly using whole blood under extreme storage and testing conditions. These data support OraQuick HCV superiority and illustrate the value of RDT evaluation under simulated field conditions.

Removal of Plasmodium falciparum-infected red blood cells from whole blood by leukoreduction filters.

BACKGROUND: There has been an unexplained decrease in the incidence of transfusion-transmitted malaria in recent years. The decrease in incidence has paralleled the increasing use of leukoreduction filters. Malaria-infected red blood cells (RBCs) share surface characteristics of hemoglobin S-containing cells. Because units collected from donors with sickle trait do not filter optimally due to adherence of RBCs to the filters, the possibility that malaria-infected RBCs may also adhere to filters was investigated. STUDY DESIGN AND METHODS: Malaria-infected whole blood or calcium ionophore (A25187)-treated and control RBCs were filtered with leukoreduction filters. Quantitation of malaria-infected RBCs before and after filtration was performed by flow cytometry to determine the presence of DNA within RBCs, indicating malaria infection. Annexin V binding was also determined before and after filtration of RBCs treated with A25187. Immediately after filtration, filters were fixed and examined by scanning and transmission electron microscopy. RESULTS: There were at least three configurations of adherence of malaria-infected RBCs demonstrated within the filters. The first was direct adherence of infected RBCs to filter fibers; the second involved adherence of malaria-infected RBCs to platelets, which were adherent to filter fibers; and the third was adherence of infected RBCs to other RBCs. Filtration also resulted in preferential removal of phosphatidylserine (PS)-expressing cells as seen by the reduction of annexin V binding after filtration. This was further confirmed by electron micrographic examination of the filters in which untreated RBCs sit within the filter resting on top of filter fibers; however, calcium ionophore-treated RBCs are seen to cling tightly to the fibers. CONCLUSIONS: PS expression by RBCs leads to their adherence within leukoreduction filters. Malaria-infected RBCs are retained via more than one mechanism. The efficiency of removal requires further study.

A Leishmania minicircle DNA footprint assay for sensitive detection and rapid speciation of clinical isolates.

BACKGROUND: Diversity in clinical outcome, due to different species of Leishmania, and its presence in asymptomatic blood donors in endemic areas warrant development of methods that are sensitive and can rapidly identify infecting species. STUDY DESIGN AND METHODS: The kinetoplast minicircle DNA is known to have heterogeneity in sequence and is present in many thousands of copies in Leishmania. Fluorescence-based polymerase chain reaction (PCR) was used to amplify minicircle DNA from six Leishmania species from different geographic locations. The sequences were then used to construct a phylogenetic tree. Speciation of 46 blinded parasite clinical isolates from various geographic regions was validated using the assay. RESULTS: Analysis displayed a distinct cluster for each species or strain. Forty-three of 46 isolates were correctly assigned to the same species identified by isoenzyme electrophoresis. The three untyped isolates were all either new species or samples from a unique geographic region. The minicircles of the three isolates formed new clusters in the tree analysis. Using minicircle DNA as PCR target, the sensitivity of the parasite detection in the spiked blood samples was five parasites per mL. CONCLUSION: Increased sensitivity and speciation without the need for parasite culture will be useful for diagnosis and treatment in clinical settings.

Transfusion-associated microchimerism in combat casualties.

BACKGROUND: Fresh whole blood (FrWB) is routinely used in the resuscitation of combat casualties in Operation Iraqi Freedom and Operation Enduring Freedom. However, studies have shown high rates (20%-40%) of transfusion-associated microchimerism (TA-MC) in civilian trauma patients receiving allogenic red blood cell (RBC) transfusions. We explored the incidence of TA-MC in combat casualties receiving FrWB compared with patients receiving standard stored RBC transfusions. METHODS: Prospective data on TA-MC at >or=14 days posttransfusion were collected on 26 severely injured combat casualties admitted to the National Naval Medical Center between December 2006 and March 2007. Demographic variables included age, sex, Injury Severity Score, and transfusion history. Data are expressed as mean +/- SD. RESULTS: The mean age of the study cohort was 24 +/- 7; mean Injury Severity Score was 17 +/- 12. All were men and suffered penetrating injury. Average hospital length of stay was 46 +/- 35 days. TA-MC was present in 45% (10 of 22) patients who were transfused at least 1 unit of blood. The four nontransfused patients all tested negative for TA-MC. Among six patients who received 4 to 43 units of FrWB, five also received RBCs and one aphaeresis platelets. The remaining 16 transfused patients who received RBCs (no FrWB) included seven who also received platelets in theater. The prevalence of TA-MC was 50% (3 of 6) in FrWB patients, 50% in patients given platelets (4 of 8), and 38% (3 of 8) in those given only RBCs as a cellular component (p = 0.61). CONCLUSIONS: Although these preliminary data do not demonstrate a significantly increased rate of TA-MC in FrWB or apheresis platelets recipients compared with RBC recipients, the overall 45% (10 of 22) rate of TA-MC in transfused soldiers warrants further study to ascertain possible clinical consequences such as graft-versus-host or autoimmune disease syndromes.

Inactivation of Leishmania donovani infantum and Trypanosoma cruzi in red cell suspensions with thiazole orange.

BACKGROUND: Methods for pathogen inactivation are currently available in some European countries for treatment of plasma and platelet (PLT) components; no approved method for treatment of red cells (RBCs) or whole blood is ready for implementation. In a previous study, thiazole orange (TO), a dye commonly used to count reticulated RBCs and PLTs, exhibited potent photoactivity against human immunodeficiency virus-1 and several model viruses in RBC suspensions. The aim of this study is to further evaluate the ability of TO to inactivate pathogens by measuring its activity against the protozoa Leishmania donovani infantum and Trypanosoma cruzi. STUDY DESIGN AND METHODS: RBC suspensions were deliberately contaminated with L. donovani infantum promastigotes or T. cruzi trypomastigotes and either maintained as an untreated control, incubated with 80 mumol per L TO in the dark, or treated with TO and light. Control and treated samples were inoculated into medium and subsequently microscopically examined for growth. RESULTS: No growth was observed in samples treated with TO in the presence or absence of light, while matched control samples lacking TO and diluted up to 5 log consistently demonstrated Leishmania or T. cruzi growth (n = 3). CONCLUSION: TO inactivated Leishmania or T. cruzi to the limit of detection in RBC suspensions without intentional illumination.

Neutrophil priming, caused by cell membranes and microvesicles in packed red blood cell units, is abrogated by leukocyte depletion at collection.

UNLABELLED: BACKGROUND AND OBJECTS: Lipids with platelet activating factor (PAF)-like activity in supernatant of packed red blood cells (PRBC) cause priming of the neutrophil respiratory burst. This effect increases with length of storage. Washing of PRBC has been considered as a means to eliminate this effect; however, the role of the cellular component was not evaluated independently of the supernatant. The source of the inflammatory lipids of the supernatant is likely to be cell membranes altered during ageing in storage and therefore, washing will not eliminate neutrophil priming caused by transfusion of aged PRBC units. The ability of washed PRBC to prime mononuclear cells for another known effect of PAF, the production of IL-8, and the probability that this lipid activity is present on microparticles in PRBC supernatant were also investigated. MATERIALS AND METHODS: At collection 10 units of whole blood were split into two equal aliquots one filtered and one unfiltered. PRBC were prepared and stored at 4 degrees C in CPD-AS5. Each week, fresh neutrophils were incubated with samples of washed PRBC and fixed. Change in CD11b, a marker known to increase on the surface of primed neutrophils, was determined by flow cytometry. To determine whether neutrophil priming ability of PRBC supernatant is contained on microvesicles, centrifuged and uncentrifuged supernatant samples were incubated with fresh neutrophils and change in CD11b expression was determined. Plasma IL-8 levels were also measured after exposure of monocytes from fresh whole blood to filtered and unfiltered washed PRBC with and without the addition of fMLP. RESULTS: Washed PRBC caused a 50-116% increase in CD11b neutrophil surface expression over baseline expression. Filtration of whole blood at collection reduced this CD11b up-regulation by 25-34%. Reduction of priming ability by filtration began on the day of collection and persisted for the storage life of the units. Centrifugation resulted in a reduction of CD11b up-regulation of 11-28% compared with unspun supernatant. Incubation of unfiltered PRBC resulted in priming of mononuclear leukocytes for IL-8 production with a 73-109% increase over baseline, but no increase over baseline was seen for incubation with filtered blood. CONCLUSION: Washing does not eliminate the ability of PRBC units to prime neutrophils and mononuclear cells, because the cellular component of PRBC, in addition to the supernatant, induces priming. Leukodepletion filters significantly reduce these effects compared with unfiltered PRBC. The in vitro beneficial effect of filtration lasts for the shelf life of 42 day units. The ability of PRBC supernatant to prime neutrophils is present on microvesicles.

Stored packed red blood cells contain a procoagulant phospholipid reducible by leukodepletion filters and washing.

BACKGROUND: Ageing RBC gradually increase the exposure of phosphatidylserine (PS) on their surface, due to loss of membrane asymmetry. PS expression on red cells is not normally a significant factor in the hemostatic process, because aged RBC are rapidly cleared from the circulation. We propose that the presence of many altered red cells during massive transfusion can lead to increased procoagulant activity similar to what is seen in disease states where it is known to play a pathophysiologic role in microvascular disease. STUDY DESIGN AND METHODS: Procoagulant activity of phospholipid generated during storage of PRBC was evaluated using PRBC's as the only source of phospholipid in the determination of modified Russell's viper venom times of 10 PRBC units in which half of each unit was left unfiltered and half of each unit filtered. Florescent labeled annexin V binding by PRBC was also assessed by flow cytometry over time in storage. The effect of washing and filtration on these parameters was also determined. RESULTS: As time of storage increased, the Russell's viper venom time of both the unfiltered and filtered units shortened (p<0.01). There was a significant lengthening of the Russell's viper venom time at all time points measured when unfiltered units were compared to filtered units (p<0.01). In both unfiltered and filtered units, with increased length of storage, there was a gradual increase in the percentage of cells or particles binding annexin V (p<0.01). Filtration resulted in a significant reduction in the percentage of cells or particles binding annexin V at all time points measured (p<0.01). The effect of washing of PRBC units on the RVVT was assessed for unfiltered and filtered units on day 42. Washing resulted in a significant reduction of the RVVT in both unfiltered and filtered groups (p<0.01). CONCLUSIONS: Levels of annexin V binding and procoagulant phospholipid activity similar to levels seen in disease states associated with significant vasoocclusive pathophysiology were found toward the end of the storage period of PRBC units. It was possible to reduce both of these parameters by leukodepletion at collection, and with washing of PRBC at the end of storage. Filtration at collection resulted in a 67% increase in RVVT over unfiltered units by day 42 of storage. On day 42 of storage, washing of filtered units resulted in a 21% increase in RVVT, and washing of unfiltered units resulted in a 34% increase in RVVT. The effects seen with filtration and washing were additive suggesting that in spite of filtration at collection, deterioration of cells continues based on age since further removal of phospholipid can be induced with washing of filtered units on day 42.

Pathogen inactivation of Trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND: Emigration of people infected with Trypanosoma cruzi to non-endemic areas has resulted in transfusion transmission to immunocompromised recipients. We studied the feasibility of inactivating T. cruzi using a new technology which utilizes riboflavin as a photosensitizer in combination with UV light, Mirasol PRT. METHODS: One billion T. cruzi organisms and 30 mL of 500 microM riboflavin were added to each of six units of human plasma and six units of platelets. To determine the level of detection of organism, a sample of each unit was cultured in tenfold serial dilutions beginning with 100 billion/250 mL as the starting culture. After 30 min, each unit was illuminated with 5.9 J/cm(2) of UV light (6.24 J/mL). The units were then cultured again in tenfold serial dilutions post-treatment. RESULTS: A 6 log reduction of pathogens was demonstrated in 5 of 6 units of plasma, and a 7 log reduction of pathogens was demonstrated in one unit. A 6 log reduction of pathogens was demonstrated in 3 of 6 units of platelets, a 7 log reduction was demonstrated in 2 of 6 units of platelets, and an 8 log reduction of pathogens was demonstrated in 1 of 6 units. CONCLUSIONS: Mirasol PRT treatment demonstrated an ability to inactivate 5-7 logs of T. cruzi in plasma and platelets.

Photoinactivation of Trypanosoma cruzi in red cell suspensions with thiopyrylium.

Chagas disease, endemic in rural areas of Mexico, Central and South America, is caused by the protozoan parasite, Trypanosma cruzi, which is spread by the Reduviid bug and also by transfusion or organ transplant. Transmission of the organism from asymptomatic donors to immunocompromised recipients, leads to clinically apparent disease. With recent immigration patterns, T. cruzi is now becoming an increasing problem in non-endemic areas of North America and Europe. Blood screening tests for T. cruzi are being developed, and one test is currently licensed by the United States Food and Drug Administration and has been implemented in some US blood centers. This study alternatively investigates the potential for a novel DNA-intercalating photosensitizer, thiopyrylium (TP), to inactivate T. cruzi in red cell suspensions. With complete inactivation using 6.3 microM of TP and 1.1J/cm(2) red light treatment, results suggest that the organism is highly sensitive to photoinactivation under conditions much less stringent than those that have been previously demonstrated to maintain red cell (RBC) properties during 42 day storage.

Leishmania: risk to the blood supply.

Leishmania infection is most often transmitted to humans via the bite of the phlebotomine sandfly, but transmission of Leishmania by transfusion has also been reported. There has been a huge increase in the incidence of cutaneous and visceral leishmaniasis in Iraq and Afghanistan. The deployment of US troops to these countries and published case reports of transmission to soldiers in endemic areas, by transfusion to infants with immature immune systems, and to individuals immunocompromised by disease or immunosuppressive therapy beckon a reexamination of blood donor deferral procedures. The length of the ongoing military conflict and the nature of exposure indicate that prior decisions regarding blood donor deferral made during the first Gulf War may no longer apply. Operation Iraqi Freedom and Operation Enduring Freedom present a much greater Leishmania threat than did Operation Desert Storm. Because most transmission by transfusion occurs in endemic areas, and visceral infection is asymptomatic in healthy individuals such as blood donors, it is difficult to determine the absolute risk of transmission by transfusion, but review of the literature provides many clues as to the appropriate measures to be taken for blood donor deferral.

Leukodepletion filters reduce Leishmania in blood products when used at collection or at the bedside.

BACKGROUND: Leishmania is an intracellular parasite of monocytes transmissible by transfusion. The feasibility of reducing Leishmania with leukodepletion filters was studied. At collection, infected blood contains the amastigote form of Leishmania within monocytes. Amastigotes cause the rupture of monocytes releasing free amastigotes that convert to promastigotes, which exist extracellularly at blood storage temperatures. Leukodepletion filters were tested at various time points in this process. STUDY DESIGN AND METHODS: Blood products were infected with Leishmania organisms and then filtered with whole-blood filters at collection, with bedside filters after storage, and to determine whether free promastigotes could be eliminated. RESULTS: Filtration at collection reduced Leishmania by 3 to 4 log or to the level of detection. Filtration of infected red cells after 2 weeks of storage showed a reduction of Leishmania by 4 log. Filtration resulted in a 6- to 8-log reduction in promastigotes either in the presence or in the absence of white cells within the filter. CONCLUSION: Filtration at the time of collection and after storage of Leishmania-infected blood resulted in a substantial reduction of free and intracellular organisms. There is currently no donor screen for Leishmania. Until adequate testing is developed, the use of leukodepletion filters could add to the safety of the blood supply.